We plan to evaluate some of the regulatory mechanisms governing contraction in vascular smooth muscle. Phosphorylation of the 20,000 dalton light chain of myosin permits rapid crossbridge cycling (shortening velocity) and thus rapid stress development. Another form of crossbridge interaction, termed "latch," allows attached crossbridges to maintain stress with greatly reduced crossbridge cycling and ATP consumption. Both forms of crossbridge interactions are dependent on (Ca2+)i. We have techniques to measure myosin phosphorylation in intact tissues and to biophysically quantitate crossbridge interactions. Correlation of these measurements with estimates of intracellular (Ca2+) in smooth muscle is the goal of this project. Estimation of intracellular free Ca2+ involves loading the photoprotein aequorin intracellularly and measuring the photon emission as aequorin binds Ca2+. We already have reported a correlation of light estimated (Ca2+)i with myosin phosphorylation in K+ depolarized swine carotid media. This correlation supports the fourth criterion of Krebs and Beavo for acceptance of Ca2+- stimulated myosin phosphorylation as physiologically significant. The (Ca2+) sensitivity for stress maintenance was higher than for myosin phosphorylation or shortening velocity. We plan to (1) further test the validity of the aequorin method by simultaneously measuring (Ca2+)i in single cells using aequorin and the fluorescent dyes. (2) Test the universality of the (Ca2+)- myosin phosphorylation and (Ca2+)-stress relationships. This involves measuring (Ca2+), myosin phosphorylation, shortening velocity, and stress during contraction with several agonists and during cyclic nucleotide-dependent vasodilation. (3) Test the effect of mechanical perturbations, which could mediate myogenic responses, on (Ca2+)i and the (Ca2+) sensitivity of the contractile apparatus.